This invention relates generally to delivery of a substance to the bloodstream of a subject, in particular to bloodstream-directed delivery of a polypeptide.
The ability to replace defective or absent genes has attracted wide attention as a method to treat a variety of human diseases (Crystal 1995 Science 270:404), Lever et al. 1995 Gene Therapy. Pearson Professional, New York p. 1-91; Friedmann 1996 Nature Med. 2:144). Although originally intended as a means of correcting inherited disorders in certain populations of somatic cells, gene-based therapy can be a useful means to supply exogenous gene products to the circulatory system for the treatment of a wide range of systemic disorders that involve deficiencies in circulating proteins, such as hormones, growth factors, and clotting proteins (Lever et al. 1995 supra; Buckel 1996 TiPS 17:450), as well as a means of administering other polypeptide drugs. The success of this application depends upon developing effective methods to both manufacture the desired protein in vivo and then secrete it into blood (Crystal 1995 supra; Lever et al. 1995 supra).
Currently, DNA-based therapy (i.e., gene therapy) is carried out in a variety of ways but involves two general protocols. In the first method, referred to as ex vivo gene therapy, cells are extracted from an individual and subjected to genetic manipulation. After genetic material has been properly inserted into the cells, the cells are implanted back into the individual from which they were removed. Persistent, in vivo expression of the newly implanted genetic material after transplantation of the transformed cells has been successful (see Morgan et al., Science 237:1476 (1987); and Gerrard et al., Nat. Genet. 3:180 (1993)). In the second approach to DNA-based therapy, referred to as in vivo gene therapy, cells within a living organism are transformed in situ with exogenous genetic material.
Several different methods for transforming cells can be used in accordance with either the ex vivo or in vivo transfection procedures. For example, various mechanical methods can be used to deliver the genetic material, including the use of fusogenic lipid vesicles (liposomes incorporating cationic lipids such as lipofection; see Felgner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987)); direct injection of DNA (Wolff, et al., Science (1990) 247:1465-1468); and pneumatic delivery of DNA-coated gold particles with a device referred to as the gene gun (Yang et al., Proc. Natl. Acad. Sci. U.S.A. 1990; 87:1568-9572). Morsy et al. reviews several of the different techniques useful in transformation of cells ex vivo or in vivo and provides citations of numerous publications in each area (Morsy et al., JAMA 270:2338-2345 (1993)).
One method of particular interest for delivery of genetic material involves use of recombinant viruses to infect cells in vivo or ex vivo. In these methods, a virus containing the desired genetic material is allowed to infect target cells within the subject. Upon infection, the virus injects its genetic material into the target cells. The genetic material is then expressed within the target cell, providing for expression of the desired genetic material. However, it would be preferable to avoid introduction of the desired genetic material by viral infection for a number of reasons. For example, viral infection results in delivery of viral DNA in addition to the desired genetic material, which may in turn result in undesirable cellular effects such as, adverse immune reactions, productive viral replication, and adverse integration events.
There is a need in the field for a method for delivery of genetic material into a cell in vivo to provide for expression of the introduced polynucleotide and secretion of the gene product it encodes into the bloodstream. The present invention addresses this problem.
The invention features methods for delivering a polypeptide to the bloodstream of a subject by introduction of a nucleic acid construct into secretory gland cells(e.g., cells of salivary gland, pancreas, or liver). In general, the method involves introduction of a nucleic acid construct into a secretory gland duct, which introduction results in expression of a gene product encoded by the introduced construct and delivery of the gene product into the bloodstream of the subject.
A primary object is to provide a method of delivering a polypeptide to the bloodstream of a subject by introducing a nucleic acid construct into cells of a secretory gland, e.g., liver, pancreatic or salivary gland (e.g., parotid gland) cells, preferably by introduction of the construct into a duct of a secretory gland. The secretory gland cells subsequently express a biologically active protein, which protein is secreted into the circulatory system.
Another object is to provide a non-invasive method of protein delivery (i.e., the method involves introduction of the nucleic acid of interest from outside the body (i.e., from the duct system of particular glands) wherein cells of a secretory gland, preferably the pancreas, salivary gland, or liver of a mammal are genetically modified to express a biologically active and therapeutically useful polypeptide, which polypeptide is secreted into the circulatory system of the individual.
Another object is to produce genetically transformed secretory gland cells which cells have incorporated into their genome genetic material which, when expressed, produces a biologically active and therapeutically useful protein which is secreted into the circulatory system.
An advantage of the present invention is that polypeptides can be delivered into the bloodstream on either a long term basis (e.g., by repeated administration of the construct) or on a short term basis. Thus, the invention is useful for treatment of diseases or conditions wherein individuals are suffering from a deficiency in a particular protein and/or can benefit from administration of an exogenous protein having a desired activity (e.g, antimicrobial activity).
Another advantage of the invention is that, in one embodiment, the nucleic acid constructs can be introduced for expression in a secretory gland cell without the need to contain the construct within a virion (e.g., the method does not require the use of a viral vector containing the construct to introduce the nucleic acid of interest into the secretory gland cell).
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the vectors, cell lines and methodology as more fully set forth below.